Sucrose Sulfhydryl group

In some instances, it might be necessary to supplement the 0.32M sucrose solution, pH 7.4, with 1 mAf EDTA, 0.25 mM dithiothreitol (DTT), and/or a cocktail of protease inhibitors. Membrane-bound phospholipases and proteases can be activated during cell disruption. EDTA chelates metal ions (calcium and magnesium) that activate certain phospholipases. DTT is a reducing agent that prevents oxidation of functionally important sulfhydryl groups. Cell disruption may also cause release of proteases from lysosomes. Protease attack on membrane proteins can be prevented by addition of protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF an inhibitor of serine proteases) and/or E-64 (an inhibitor of cysteine proteases), to the sucrose solution... 

MSAS from P. patulum was separated from the FAS via sucrose gradient centrifugation [121,122] and thus shown to constitute a distinct multifunctional enzymatic system. It was purified to homogeneity and found to be a 190 kDa multifunctional enzyme [22,120]. The enzyme was more stable in the presence of its substrates and at mildly basic pH values. The pH optimum of the enzyme was 7.6 and apparent K values for its substrates were 10 pM (acetyl-CoA), 7 pM (malonyl CoA), and 12 pM (NADPH) [115,120,123]. The rate for triacetate lactone formation in the absence of NADPH was determined to be ten-fold lower than for 6-MSA formation (Fig. 5) [120]. Analogous to FASs and peptide synthetases, 4 -phosphopantetheine is a covalently bound cofactor of 6-MSAS [124]. Likewise, iodoacetamide and N-ethylmaleimide were found to inactivate the enzyme, suggesting the presence of catalytic sulfhydryl residues in 6-MSAS [124]. Furthermore, in the presence of malonyl CoA and NADPH, low concentrations of iodoacetamide convert 6-MSAS into a malonyl CoA decarboxylase. Without external addition of acetyl-CoA, 6-MSAS decarboxylates the malonyl group and the derived acetyl moiety is used as a starter unit for the formation of 6-MSA [125]. 

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